This invention relates to a method for the determination of creatine phosphokinase.
Creatine phosphokinase (CPK) is a kinase enzyme which catalyzes the reversible transfer of a phosphate group from creatine phosphate to adensoine-5'-diphosphate (ADP) according to the following equation: ##EQU1##
CPK activity is greatest in striated muscle tissue, brain and heart tissue. Serum CPK activity is elevated in all types of muscular dystrophy and becomes elevated within a few hours after a myocardial infarction. Consequently, tests for CPK activity levels are of significant clinical interest.
Various methods have been developed heretofore for the assay of CPK, including colorimetric, fluorimetric, and coupled enzymatic methods.
In one typical coupled enzyme system, the reaction of creatine and ATP is initially catalyzed by CPK to form creatine phosphate and ADP. This reaction is then coupled to two other enzyme reactions which employ phosphoenolpyruvate, reduced nicotinamide adenine dinucleotide (NADH) and the enzymes pyruvic kinase and lactate dehydrogenase. These reactions lead ultimately to the oxidation of NADH which is followed spectrophotometrically at 340 nm. This method was developed essentially by Tanzer and Gilvarg, J. Biol. Chem. 234, 3201-4 (1959), and modifications are described in U.S. Pat. No. 3,403,077.
Another coupled enzyme method is based on the reverse reaction in which creatine phosphate and ADP substrates react in the presence of CPK to form creatine and ATP. The ATP generated serves in an auxiliary reaction to phosphorylate glucose in the presence of hexokinase (HK). The resulting glucose-6-phosphate (G-6-P) then becomes a substrate for the ultimate indicator reaction which is catalyzed by glucose-6-phosphodehydrogenase (G-6-PDH) in the presence of nicotinamide adenine dinucleotide phosphate (NADP) to form 6-phosphogluconate and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The production of NADPH is followed spectrophotometrically at 340 nm. This coupled enzyme system can be shown by the following series of equations: ##EQU2##
The latter coupled enzyme system, first described by Nielsen and Ludvigsen and by Oliver, has been amplified by Rosalki, J. Lab. Clin. Med. 69, 696-705 (1967), and further modifications are disclosed in U.S. Pat. Nos. 3,413,198, 3,485,724 and 3,540,984.
While the foregoing spectrophotometric methods are useful, they have the disadvantage in that each individual serum sample assay takes at least about 8 to 15 minutes to complete.